Project Abstract Ubiquitination is an essential post-translational modification that regulates nearly all cellular processes, including cell division, differentiation, and protein quality control. As a result, aberrant ubiquitin signaling gives rise to diseases such as cancer and neurodegeneration. Ubiquitination is carried out by a trio of enzymes (E1, E2, E3), including a diverse set of E3 ligases that catalyze the direct (RING) or indirect (HECT) transfer of ubiquitin to substrates. Recently, the Klevit laboratory classified the RING-Between-RING (RBR) family of E3s as a unique RING/HECT hybrid, in which a RING domain recruits the E2, but subsequent catalysis is performed through HECT-like cysteine intermediates. Dysfunction of RBR enzymes has been implicated in neurodegeneration, defects in organogenesis, and inflammation, yet the molecular mechanisms surrounding their unique catalysis and substrate targeting remains to be characterized. HHARI is a member of the auto- inhibited Ariadne family of RBR ligases, suggesting its temporal activation has important cellular consequences. While RBR enzymes share a common catalytic core, nothing is known about how they recognize their substrates. Recent structural studies of inhibited HHARI reveal significant separation between domains involved in E2 binding and catalysis. Given these results, we hypothesize that RBR enzymes contain unique surfaces involved in substrate recruitment and require large conformational changes to orient active sites for catalysis. To investigate this, I will carry out the following aims: Aim 1: How does HHARI recognize its substrate 4EHP. To understand how and where substrates bind, I will use high- and low- resolution structural techniques to define the interaction surface(s) on both HHARI and 4EHP. Whether these binding events contribute to global conformational changes will be explored using SAXS and HDX analysis. Additionally, these studies will take advantage of strategies used to trap ubiquitin intermediates, allowing for the study of distinct transition steps. The interactions identified through these studies will provide a foundation for understanding how RBRs select and engage their substrates. Aim 2: Functional characterization of the HHARI homolog, ARI-1, in C. elegans. Using the C. elegans homologs, ARI-1 (HHARI) and IFE-4 (4EHP), I will investigate the functional importance of RBR substrate recognition and catalysis in vivo. I will use CRISPR/Cas9 gene editing to identify residues critical for distinct RBR functions, as well as substrate interactions with IFE-4. The functional consequences of these mutations will be evaluated by immunoprecipitation (IP), microscopy, and phenotypic characterization. Additionally, IP and mass-spectrometry will be used to identify an ARI-1 interaction network, which is likely to include novel substrates and provide insight into associated cellular pathways. Given our limited understanding of HHARI activation in vivo, these studies will provide insight into the temporal control and cellular consequences of HHARI activity, and will elucidate mechanisms controlling RBR substrate selection that could be exploited for therapeutic interventions.